Dihydrofolate reductase from an amethopterin-resistant strain of Streptococcus faecium contains nine tryptophan residues and one cysteine residue per mole of protein (mol. wt. 20,000). N-Bromosuccinimide treatment of the enzyme leads to decreases in enzyme activity. Complete loss of activity corresponds to the oxidation of two tryptophan residues. Other residues amenable to attack by the oxidant are not altered. The oxidation of these two critical tryptophans drastically reduces the aromatic side-chain Cotton effect of the native enzyme in the 260 to 295 nm region of its circular dichroic absorption spectrum. The evidence indicates that each of these two critical tryptophans contributes equally to the aromatic side-chain Cotton effect in this spectral region.